CN107446998A - MTHFR C677T, rs1801133 single nucleotide polymorphisms quick detections primer, molecular beacon, kit and its detection method - Google Patents
MTHFR C677T, rs1801133 single nucleotide polymorphisms quick detections primer, molecular beacon, kit and its detection method Download PDFInfo
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Abstract
The invention discloses MTHFR C677T, the kit of RS1801133 gene pleiomorphism quick detections, including PCR reaction solutions；The raw material of PCR reaction solutions and final concentration of：The U/ μ L of archaeal dna polymerase 2.05；dNTPs 0.1‑0.5mM；The X of 5X PCR buffer solutions 0.5 1.5；MgCl2 1‑2.5mM；Lauryl sodium sulfate 0.0005 0.015%（w/v）；Triton X-100 0.001 0.03%（w/v）；0.2 0.7 μM of sense primer；0.2 0.7 μM of anti-sense primer；0.2 0.7 μM of mutant molecules beacon；0.2 0.7 μM of wild type molecular beacon；And it is used to detect cell sample.The technical problem to be solved in the present invention is to provide it is a kind of it is simple to operate, avoid polluting and detect quick MTHFR C677T, primer, molecular beacon, kit and its detection method involved by RS1801133 gene pleiomorphism quick detections.
Description
Technical field
The present invention relates to biology field, and in particular to a kind of MTHFR-C677T, RS1801133 gene pleiomorphism
Primer, molecular beacon, kit and its detection method of quick detection.
Background technology
SNP(single nucleotide polymorphism)：The single core of genomic dna sequence same position between individual
The caused polymorphism of thuja acid variation.The polymorphism that SNP is showed relates only to the variation of single base, can be turned by single base
Change, transversion, insertion or missing cause, and frequency of any allele in colony be not less than 1%.In general,
One SNP site only has two kinds of allele, therefore is called diallele.SNP is the third generation point after RFLP and STR
Son mark, have the characteristics that quantity is more, distribution is wide and inheritance stability.It is directly related with many diseases, is to determine human diseases
The principal element of neurological susceptibility and drug response difference.Therefore, snp analysis for Population Genetics, disease related gene research,
The field important roles such as new drug research, clinical examination and molecule diagnosis.
Molecular beacon (molecular beacon) is that a kind of one kind based on the design of FRET principle is new
Type nucleic acid fluorescent probe, it is combined the change of meeting recurring structure with target sequence so as to influence fluorescence signal.It is a kind of last 5 ' and 3 '
Hold and itself form the stem ring double labelling oligonucleotide probe of the hairpin structure of 8 bases or so, the nucleotide sequence at both ends is mutual
Recruit pair, therefore mark fluorophor to be at one end close to quenching group of the mark in the other end.Under this configuration,
Fluorophor after being excited caused photon be quenched agent and be quenched, so fluorescence will not be produced.In high-temperature denatured or moving back afterwards
It is this destructurized in fiery crossover process, cause fluorophor away from quenching group, fluorescence just can be excited and be monitored instrument
Device detects.
Folic acid (folic acid) be also FA, is a kind of water soluble vitamin.Folic acid is that human body is utilizing sugar
It is material necessary to body cell growth and breeding with necessary material during amino acid.Folic acid is with tetrahydrofolic acid in vivo
Form works, and tetrahydrofolic acid participates in the synthesis and conversion of purine nucleic acid and pyrimidine nucleotide in vivo.Folic acid is in manufacture nucleic acid
Play the part of important role on (ribonucleic acid, DNA).Folic acid helps the metabolism of protein, and is total to vitamin B12
It is the manufacture indispensable material of red blood cell with the generation and maturation for promoting red blood cell.Merisis and core of the folic acid to cell
The synthesis of acid, amino acid, protein plays an important role.Human body lacks the exception that folic acid can cause red blood cell, and prematurity is thin
The increase of born of the same parents, anaemia and white blood cell are reduced.Folic acid is the indispensable nutrient of embryo growth and development.Pregnant woman, which lacks folic acid, to be had
It may cause occur under-weight, harelip, heart defect etc. during fetal birth.If pregnancy first 3 months in lack folic acid, can
Cause fetal neural tube developmental defect, and cause deformity.Methylenetetrahydrofolate reductase (Methylenetetrahydrofol
Ate reductase, MTHFR) it is key enzyme in methionine-folic acid metabolism system, together with MTRR, maintain folic acid normal
Metabolism.In addition, also participate in maintaining normal homocysteine level in vivo.MTHFR and MTRR gene mutations can cause its volume
The folic acid metabolism key enzyme activity of code reduces, and causes folate metabolism disorder, causes folate level reduction and homocysteine
Mass formed by blood stasis.For the people that mthfr gene is abnormal, MTHFR enzymatic activitys substantially reduce, and cause folate metabolism disorder, cause neonate refreshing
Onset risk through diseases such as defective tube, Down's syndrome and harelips substantially increases, and this kind of people requires supplementation with more folic acid
It can be only achieved expected effect.The most common two SNP 677C of MTHFR>T and 1298A>C, in Chinese population
In incidence be about 45.2% and 18.6%.Mutational site common MTRR is 66A>G, the incidence in Chinese population is about
For 25.7%., can be to personal folic acid metabolism energy by being detected to folic acid metabolism relevant enzyme MTHFR and MTRR gene loci
Power is assessed, can be with adjuvant clinical doctor to pregnant woman's expected date of childbirth neonate NTD and cardiovascular disease incidence risk
Assessed, so as to instruct folic acid individuation to use.
MTHFR (C677T, rs1801133) wild type genotype is C/C, and its mutated-genotype has heterozygote C/T and homozygosis
Sub- two kinds of T/T.Metabolism of the wild type C/C patient to medicine belongs to eubolism type, metabolism of the heterozygote C/T patient to medicine
Type belongs to medium metabolic type, and homozygote T/T patient belongs to slow inactivation to the metabolic type of medicine.By to MTHFR
(C677T) detection of gene loci, show which kind of genotype measured is, so as to instruct the accurate medication of carry out of doctor as early as possible,
Improve medicine and use validity, reduce toxic side effect.
Nowadays, the detection product of folic acid metabolism enzyme gene site (MTHFR and MTRR) mainly passes through PCR- chip hybridizations
Method, fluorescent PCR method or PCR sequencing PCR etc. are realized, but these technologies need to extract the blood of measured, then extract DNA etc., from
Draw blood at least needs 4 hours to extraction purification DNA, and whole detection process will at least take more than 8 hours, and these methods have
There are the invasive, complex operation of pain, clean environment, professional operation, the features such as speed is slow, the time is long.
The content of the invention
The technical problem to be solved in the present invention is to provide one kind to be sampled under painless environment, simple to operate, cost it is low and
Detect primer, the molecule letter involved by MTHFR-C677T, RS1801133 gene pleiomorphism quick detection quick, that the time is short
Mark, kit and its detection method, so as to provide guidance with folic acid for individuation, reduce fetal anomaly risk.
In order to solve the above-mentioned technical problem, the present invention provides following technical scheme：MTHFR-C677T, RS1801133 gene
The kit of polymorphism quick detection,
Including PCR reaction solutions；The raw material of PCR reaction solutions and final concentration of,
Archaeal dna polymerase 2.05U/ μ L；
dNTPs 0.1-0.5mM；
5X PCR buffer solutions 0.5-1.5X；
MgCl2 1-2.5mM；
Lauryl sodium sulfate 0.0005-0.015% (w/v)；
Triton X-100 0.001-0.03% (w/v)；
0.2-0.7 μM of sense primer；
0.2-0.7 μM of anti-sense primer；
0.2-0.7 μM of mutant molecules beacon；
0.2-0.7 μM of wild type molecular beacon；
And it is used to detect cell sample.
Using the kit of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection of technical solution of the present invention,
Using cell as sample, based on fluorescent molecular bacon method, with reference to cell pyrolysis liquid, (lauryl sodium sulfate, polyethylene glycol are pungent
Base phenyl ether) MTHFR-C677T, RS1801133 are tested and analyzed.With cell pyrolysis liquid Direct Pyrolysis during the course of the reaction
Cell simultaneously discharges DNA in nucleus.In existing PCR reactions, reacted with PCR of the cell directly as template, usual nothing
The thorough cell lysis of method, and crack after cell fragment and some cellular contents (such as protease) to PCR reaction have suppression,
It is unstable to ultimately result in genotyping result, or even failure.Therefore, not plus in the case of lysate, cell is directly added into as mould
Plate, can cause cell cracking it is not thorough, and cell cracking after cell fragment and some protease PCR can be reacted
Cause to hinder, cause the failure of an experiment.The cell pyrolysis liquid of the present invention can directly dissolve cytoplasm and cell membrane, be permitted between saboteur
More faint chemical bond, some protease are decomposed, reduce the cell cracking influence that caused impurity is reacted PCR afterwards.Therefore
The part inhibition that other compositions are reacted PCR in cell is also eliminated while maximum released dna.
From experiment, the final concentration scope of the raw materials of PCR reaction solutions, specific primer and molecular beacon is above range
When testing result it is correct.Wherein, 5X reaction buffers 1X raw material is 5X reaction buffers, and it is 5 times to refer to initial concentration
Reaction buffer, the final concentration for the application that 1X refers to.
The present invention directly scrapes oral mucosa cast-off cells with that need not take blood sample, painless noninvasive；Without specialty
Personnel, easy to operate, simple training, conventional environment, bed side samples, convenient and swift；The advantages that going out result after about 1 hour.For
Also can be that doctor as early as possible race against time by accurate medication while measured reduces painful.
Further, the raw material of described PCR reaction solutions is final concentration of：
Archaeal dna polymerase 2.05U/ μ L；
dNTPs 0.2mM；
5X PCR buffer solutions 1.1X；
MgCl22.5mM；
Lauryl sodium sulfate 0.005% (w/v)；
Triton X-100 0.01% (w/v)；
The final concentration of sense primer and anti-sense primer is 0.7 μM；
Final concentration of 0.7 μM of mutant molecules beacon；
Final concentration of 0.6 μM of wild type molecular beacon.
From experiment, testing result is most accurate when the raw material final concentration scope of PCR reaction solutions is above range, success rate
Highest.
Further, described cell sample is mucous membrane of mouth cast-off cells.The present invention is taken off with the mucous membrane of mouth directly scraped
It is sample to fall cell, easy to operate.
Further, human genome DNA's gene order of purification is also included.Kit includes quality-control product, for purification
Human genome DNA's gene order, be measure when in order to do parallel test, to determine the validity of reagent result.
The application also proposes another technical scheme, MTHFR-C677T, RS1801133 gene in kit of the present invention
The primer of polymorphism quick detection,
The gene order of sense primer 5 ' -3 ' is：AAGCACTTGAAGGAGAAGGT；
The gene order of anti-sense primer 5 ' -3 ' is：AAAGCGGAAGAATGTGTCAG.
The application also proposes another technical scheme, MTHFR-C677T, RS1801133 base of primer detection in kit
Because of the molecular beacon of polymorphism quick detection,
The gene order of the 5 ' of wild type molecular beacon -3 ' is：CGTGCAGAAATCGGCTCCCGCAGTGCACG；
5 ' -3 ' gene order of mutant molecules beacon is：CGCGACGAAATCG+ACTCCCGCAGACGTCGCG；
+ the base modified for lock nucleic acid；
And 5 ' the ends or 3 ' ends of wild type molecular beacon and mutant molecules beacon gene order are respectively equipped with fluorophor
With the quenching group coordinated with fluorophor.
The technical program has used molecular beacon molecular beacon, and molecular beacon itself can be formed to be a kind of in 5 ' and 3 ' ends
By the double labelling oligonucleotide molecules beacon of the hairpin structure of 5-8 base composition.The gene order of as above-mentioned molecular beacon
And structure.During free state, two ends of hairpin structure make fluorescence molecule with quencher molecule close to (about 7-10nm).Now
Generation FRET, the fluorescence for sending fluorescence molecule are quenched molecule absorption and distributed in the form of heat, fluorescence
Almost it is quenched completely.When the target sequence of molecular beacon and the gene order complete complementary of cell combines to form double-stranded hybrid
When, the base-pair of stem structure complementary region is separated, and distance increases between fluorescence molecule and quencher molecule, and at this moment molecular beacon is glimmering
Light almost 100% recovery.This design can effectively increase the specificity of molecular beacon, improve to MTHFR-C677T,
The correctness of RS1801133 detections.
The principle of detection is：The conservative region design pair of primers in MTHFR (C677T) gene mutation sites both sides, and
Two molecular beacons, MTHFR (C677T) mutant molecules beacon, MTHFR (C677T) wild type are designed at the sequence of mutational site
Molecular beacon, MTHFR (C677T) wild type molecular beacons are combined with unmutated sequence, MTHFR (C677T) mutant molecules letter
Mark is combined with mutational site sequence.Wild type molecular beacon, the end of mutant molecules beacon 5 ' are marked with fluorescein, and 3 ' ends are with being quenched
Group marks.Then, by scraping oral mucosa cast-off cells, it is put into detection reagent, reagent is put into qPCR instrument, and is transported
Performing PCR program, due to containing cell pyrolysis liquid in PCR reaction mixtures, cell pyrolysis liquid can fully crack mucous membrane of mouth and take off
Fall cell, and released dna, performing PCR amplification is entered in the presence of primer and archaeal dna polymerase etc., in PCR annealing stages, wild type
Molecular beacon is combined with unmutated sequence, and mutant molecules beacon is combined with the sequence for having mutational site, with reference to rear, fluorescence
Group away from quenching group, by real time fluorescent quantitative detector recorded by the fluorescence now sent, so as to pass through the face of fluorescence
Color can determine whether out to detect the genotype of gene.
Further, described fluorophor is 6-Fam labelling groups, and described quenching group is BHQ1 labelling groups；
Or fluorophor is the labelling groups of Alexa Fluor 594, described quenching group is BHQ2 labelling groups.For
Two kinds of fluorophors and quenching group used in the present invention, certainly, it is also possible to other fluorophors and quenched with what it coordinated
The group that goes out replaces, and does not influence testing result.
The application also proposes another technical scheme, is carried out using above-mentioned primer, molecular beacon and kit
MTHFR-C677T, RS1801133 gene pleiomorphism quick determination method, operating method be,
(1) gargled 2 times with water before sampling, be more than 5 seconds every time, swallowed 2-3 times after gargling；
(2) cheek wall in the nearly 90 ° of contacts oral cavity in sampling portion of sampler is taken, uniformly scrapes 5 times, is 1 time up and down；
(3) by the sampling portion insertion PCR reaction solutions of sampler after sampling, mix；
(4) human genome DNA for taking 1 μ L to purify is added in another PCR reaction solution, is mixed；
(5) the PCR reaction solutions for completing sample-adding are put into quantitative PCR apparatus, run response procedures.
Further, the response procedures of quantitative PCR apparatus are：
A, pre-degeneration：95 DEG C, 5min, 1 circulations of temperature；
B, it is denatured：95 DEG C, 8s；
C, annealing and extension：58 DEG C, 35s；
D, B and c program operate 50 circulations.
Further, described quantitative PCR apparatus is binary channels, and excitation wavelength is respectively 450-500nm and 550-600nm.
Brief description of the drawings
Fig. 1 is the action principle figure of molecular beacon of the present invention；
Fig. 2 is first sample detection amplification curve diagram of the embodiment of the present invention one；
Fig. 3 is second sample detection amplification curve diagram of the embodiment of the present invention one；
Fig. 4 is the 3rd sample detection amplification curve diagram of the embodiment of the present invention one；
Fig. 5 is the detection amplification curve diagram of the quality-control product of the embodiment of the present invention one；
Fig. 6 is first sample detection amplification curve diagram of the embodiment of the present invention two；
Fig. 7 is second sample detection amplification curve diagram of the embodiment of the present invention two；
Fig. 8 is the 3rd sample detection amplification curve diagram of the embodiment of the present invention two；
Fig. 9 is the detection amplification curve diagram of the quality-control product of the embodiment of the present invention two；
Figure 10 is first sample detection amplification curve diagram of the embodiment of the present invention three；
Figure 11 is second sample detection amplification curve diagram of the embodiment of the present invention three；
Figure 12 is the 3rd sample detection amplification curve diagram of the embodiment of the present invention three；
Figure 13 is the detection amplification curve diagram of the quality-control product of the embodiment of the present invention three；
Figure 14 is first sample detection amplification curve diagram of the embodiment of the present invention four；
Figure 15 is second sample detection amplification curve diagram of the embodiment of the present invention four；
Figure 16 is the 3rd sample detection amplification curve diagram of the embodiment of the present invention four；
Figure 17 is the detection amplification curve diagram of the quality-control product of the embodiment of the present invention four.
Embodiment
It is each embodiment in MTHFR-C677T, RS1801133 gene pleiomorphism quick detection kit of the present invention below
Raw material final concentration, embodiment is as shown in table 1：
Table 1
It is the concrete operations mode of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection of the present invention below：
First, the preparation of cell pyrolysis liquid
Cell pyrolysis liquid concentration of component in table 1 is the ultimate density in PCR reaction systems, and this concentration is too small, can cause
The inaccuracy of sample-adding.Therefore in configuration, final concentration can be expanded, then dilute.
The superclean bench of all preparation of reagents after sterilization is carried out.
(1) in embodiment one 200X cell pyrolysis liquids preparation
The collocation method of lysate：Using 1.5ml centrifuge tube, 10ul lauryl sodium sulfate SDS and 1.88ul are added
Triton X-100 Triton X-100, the water of 988.12ul nuclease frees is added to cumulative volume 1000ul, makes SDS
Final concentration reach 0.1%, TritonX-100 final concentration is reached 0.2%, as 200x cell pyrolysis liquid, Ran Houzhen
Swing mixing, -4 DEG C of preservations.
(2) in embodiment two, example IV 200X cell pyrolysis liquids preparation
The collocation method of lysate：Using 1.5ml centrifuge tube, 100ulSDS and 18.78ulTriton X-100 are added,
The water of 881.22ul nuclease frees is added to cumulative volume 1000ul, SDS final concentration is reached 1%, makes TritonX-100
Final concentration reach 2%, as 200x cell pyrolysis liquid, then concussion mix, -4 DEG C preservation.
(3) in embodiment three 200X cell pyrolysis liquids preparation
Using 1.5ml centrifuge tube, add 300ul SDS and 56.34ulTriton X-100, add 643.66ul without
The water of nuclease makes SDS final concentration reach 3%, TritonX-100 final concentration is reached 6%, i.e., to cumulative volume 1000ul
For 200x cell pyrolysis liquid, then concussion mixes, -4 DEG C of preservations.
The gene order of sense primer 5 ' -3 ' is：AAGCACTTGAAGGAGAAGGT；
The gene order of anti-sense primer 5 ' -3 ' is：AAAGCGGAAGAATGTGTCAG.
The gene order of wild type molecular beacon 5 ' -3 ' is：
6-Fam marks-CGTGCAGAAATCGGCTCCCGCAGTGCACG-BHQ1 is marked；
The gene order of mutant molecules beacon 5 ' -3 ' is：
Alexa Fluor 594 mark-CGCGACGAAATCG+ACTCCCGCAGACGTCGCG-BHQ2 to mark；
+ the base modified for lock nucleic acid.
Above-mentioned raw materials, in addition to specific primer, molecular beacon, cell pyrolysis liquid, it is purchased from Shanghai Pu Luomaige biology systems
Product Co., Ltd.Specific primer is synthesized by Shanghai Zhan Biao bio tech ltd；Molecular beacon accounts for the biological skill of mark by Shanghai
Art Co., Ltd synthesizes.
Two .PCR reaction solutions are prepared
The reaction solution of embodiment one, two, three, four prepares the reaction solution preparation such as table that information is as shown in table 2, and quality-control product is tested
Shown in 3.The superclean bench of all preparations after sterilization is carried out.
Table 2
Table 3
3rd, MTHFR-C677T, RS1801133 genotype detection；
Each embodiment respectively prepares 9 reagents and a quality-control product reagent according to 23.5ul reaction system, each tested
Person detects 3 repetitions (a by-reaction liquid), and MTHFR-C677T, RS1801133 phenotype of the quality-control product are CT.Above institute
The final concentration of finger refers to concentration of each component in 23.5ul systems, and example IV is the repetition to embodiment two, preparation of reagents
After the completion of -20 DEG C be kept in dark place；
(1) gargled 2 times with clear water before sampling, be no less than 5 seconds every time, swallowed 2-3 times after gargling, avoid oral cavity wall as far as possible
Remain saliva；
(2) disposable sterile swab (license notification number is taken out：CN205359515U) and reaction solution, pull out anti-
Answer liquid plug and swab end cap；
(3) cheek wall in the nearly 90 ° of contacts oral cavity in swab end is made, it (is up and down 1 that embodiment one, two, three, four, which uniformly scrapes 5 times,
It is secondary), dynamics is advisable with micro- dash forward of cheek；
(4) immediately by swab intercalation reaction liquid after sampling, pressing top makes itself and the reagent seal of tube, and lucifuge is kept in, and
After flick Reagent Tube and sample is fully mixed with reaction solution；
Operation is completed in step (1)~(4) in 20s, is sure not to interrupt；
(5) take 1 μ L quality-control products to be added in another by-reaction liquid with pipettor, flick Reagent Tube and mix；
(6) reaction solution for completing sample-adding is put into JY-1000A type fluorescent PCR instrument (license notification numbers：
CN103820306B in), and the Program of table 4.
Table 4
(7) amplification curve and fluorescence color, analysis result are obtained.
4th, interpretation of result：
Because human genome is diploid, two allele are contained on each gene locus.As a result three kinds are shared, point
Wei not " wild CC ", " heterozygosis CT ", " mutation T T ".Wherein negative findings is " CC ", and positive findings is " CT ", " TT ".
Accompanying drawing 1 is molecular beacon action principle figure；
The result of embodiment one is：
From accompanying drawing 2：Only C lines have exponential increase, and T lines do not have exponential increase, therefore testing result is wild type
CC, prompt to detect gene expression or activity may be normal, the genotype is eubolism type；
From accompanying drawing 3：C lines and T lines have exponential increase, represent that testing result is heterozygous variance type CT, prompt to be examined
Cls gene activity may decline, and the genotype belongs to medium metabolic type；
From accompanying drawing 4：Only T lines have exponential increase, and C lines do not have exponential increase, represent that testing result is homozygous change
Special-shaped TT, prompting detect gene activity and are decreased obviously or may lose, and its activity decrease or forfeiture may cause folic acid metabolism
It is low；
From accompanying drawing 5：C lines and T lines have an exponential increase, represent that testing result is heterozygous variance type CT, the genotype
Belong to medium metabolic type, show that the phenotype of detected quality-control product is correct；
Thus, the genotype for drawing MTHFR (C677T, rs1801133) can be analyzed from Fig. 2, Fig. 3 and Fig. 4, so as to
Draw, metabolic type of the patient to folic acid.
The result of embodiment two is：
From accompanying drawing 6：Only C lines have exponential increase, and T lines do not have exponential increase, therefore testing result is wild type
CC, prompt to detect gene expression or activity may be normal, the genotype is eubolism type；
From accompanying drawing 7：C lines and T lines have exponential increase, represent that testing result is heterozygous variance type CT, prompt to be examined
Cls gene activity may decline, and the genotype belongs to medium metabolic type；
From accompanying drawing 8：Only T lines have exponential increase, and C lines do not have exponential increase, represent that testing result is homozygous change
Special-shaped TT, prompting detect gene activity and are decreased obviously or may lose, and its activity decrease or forfeiture may cause folic acid metabolism
It is low；
From accompanying drawing 9：C lines and T lines have an exponential increase, represent that testing result is heterozygous variance type CT, the genotype
Belong to medium metabolic type, show that the phenotype of detected quality-control product is correct；
Thus, the genotype for drawing MTHFR (C677T, rs1801133) can be analyzed from accompanying drawing 6, accompanying drawing 7 and accompanying drawing 8,
So as to draw, metabolic type of the patient to folic acid.
The result of embodiment three is：
From accompanying drawing 10：Only C lines have exponential increase, and T lines do not have exponential increase, therefore testing result is wild type
CC, prompt to detect gene expression or activity may be normal, the genotype is eubolism type；
From accompanying drawing 11：C lines and T lines have exponential increase, represent that testing result is heterozygous variance type CT, prompt to be examined
Cls gene activity may decline, and the genotype belongs to medium metabolic type；
From accompanying drawing 12：Only T lines have exponential increase, and C lines do not have exponential increase, represent that testing result is homozygous change
Special-shaped TT, prompting detect gene activity and are decreased obviously or may lose, and its activity decrease or forfeiture may cause folic acid metabolism
It is low；
From accompanying drawing 13：C lines and T lines have an exponential increase, represent that testing result is heterozygous variance type CT, the genotype
Belong to medium metabolic type, show that the phenotype of detected quality-control product is correct；
Thus, the gene for drawing MTHFR (C677T, rs1801133) can be analyzed from accompanying drawing 10, accompanying drawing 11 and accompanying drawing 12
Type, so as to draw, metabolic type of the patient to folic acid.
Can be seen that by above three embodiment result, three embodiments can Correct Analysis go out MTHFR (C677T,
Rs1801133 genotype), but three kinds of genotype fluorescent values are significantly better than that embodiment one and embodiment three in embodiment two.
Therefore determining that the PCR reaction systems in embodiment two are optimal, the later stage is repeated once to embodiment two again, as example IV.
The result of example IV is：
From accompanying drawing 14：Only C lines have exponential increase, and T lines do not have exponential increase, therefore testing result is wild type
CC, prompt to detect gene expression or activity may be normal, the genotype is eubolism type；
From accompanying drawing 15：C lines and T lines have exponential increase, represent that testing result is heterozygous variance type CT, prompt to be examined
Cls gene activity may decline, and the genotype belongs to medium metabolic type；
From accompanying drawing 16：Only T lines have exponential increase, and C lines do not have exponential increase, represent that testing result is homozygous change
Special-shaped TT, prompting detect gene activity and are decreased obviously or may lose, and its activity decrease or forfeiture may cause folic acid metabolism
It is low；
From accompanying drawing 17：C lines and T lines have an exponential increase, represent that testing result is heterozygous variance type CT, the genotype
Belong to medium metabolic type, show that the phenotype of detected quality-control product is correct；
Thus, the gene for drawing MTHFR (C677T, rs1801133) can be analyzed from accompanying drawing 14, accompanying drawing 15 and accompanying drawing 16
Type, so as to draw metabolic type of the patient to folic acid, its result is reliable and stable, success rate 100%.
Nucleotides sequence list is as follows：
<110>Chongqing Jing Yin biotechnologies Co., Ltd
<120>The primer of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection, molecular beacon, kit and
Its detection method
<160>4
<210>1
<211>20
<212>DNA
<213>Artificial sequence
<220>
<221>prim_bind
<400>1
AAGCACTTGAAGGAGAAGGT
<210>2
<211>20
<212>DNA
<213>Artificial sequence
<220>
<221>prim_bind
<400>2
AAAGCGGAAGAATGTGTCAG
<210>3
<211>29
<212>RNA
<213>Artificial sequence
<220>
<221misc_binding
<400>3
CGTGCAGAAATCGGCTCCCGCAGTGCACG
<210>4
<211>32
<212>DNA
<213>Artificial sequence
<220>
<221>misc_binding
<400>4
CGCGACGAAATCG+ACTCCCGCAGACGTCGCG。
For those skilled in the art, on the premise of technical solution of the present invention is not departed from, if can also make
Dry modification and improvement, these should also be considered as protection scope of the present invention, these all without influence effect that the present invention implemented and
Practical applicability.
<110>Chongqing Jing Yin biotechnologies Co., Ltd
<120>Primer, molecular beacon, kit and its inspection of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection
Survey method
<160>4
<210>1
<211>20
<212>DNA
<213>Artificial sequence
<220>
<221>prim_bind
<400>1
AAGCACTTGAAGGAGAAGGT
<210>2
<211>20
<212>DNA
<213>Artificial sequence
<220>
<221>prim_bind
<400>2
AAAGCGGAAGAATGTGTCAG
<210>3
<211>29
<212>RNA
<213>Artificial sequence
<220>
<221> misc_binding
<400>3
CGTGCAGAAATCGGCTCCCGCAGTGCACG
<210>4
<211>32
<212>DNA
<213>Artificial sequence
<220>
<221> misc_binding
<400>4
CGCGACGAAATCG+ACTCCCGCAGACGTCGCG。
Claims (10)
1.MTHFR-C677T, the kit of RS1801133 gene pleiomorphism quick detections, it is characterised in that including PCR react
Liquid；The raw material of PCR reaction solutions and final concentration of,
The U/ μ L of archaeal dna polymerase 2.05；
dNTPs 0.1-0.5mM；
5X PCR buffer solution 0.5-1.5 X；
MgCl2 1-2.5mM；
Lauryl sodium sulfate 0.0005-0.015%（w/v）；
Triton X-100 0.001-0.03%（w/v）；
0.2-0.7 μM of sense primer；
0.2-0.7 μM of anti-sense primer；
0.2-0.7 μM of mutant molecules beacon；
0.2-0.7 μM of wild type molecular beacon；
And it is used to detect cell sample.
2. the kit of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection according to claim 2, it is special
Sign is：The raw material of described PCR reaction solutions is final concentration of：
The U/ μ L of archaeal dna polymerase 2.05；
dNTPs 0.2mM；
5X PCR buffer solutions 1.1X；
MgCl2 2.5mM；
Lauryl sodium sulfate 0.005%（w/v）；
Triton X-100 0.01%（w/v）；
The final concentration of sense primer and anti-sense primer is 0.7 μM；
Final concentration of 0.7 μM of mutant molecules beacon；
Final concentration of 0.6 μM of wild type molecular beacon.
3. the kit of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection according to claim 3, it is special
Sign is：Described cell sample is mucous membrane of mouth cast-off cells.
4. the kit of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection according to claim 4, it is special
Sign is：Also include the human genome DNA of purification.
5. MTHFR-C677T, RS1801133 gene pleiomorphism in the kit according to claim 1 ~ 4 any one
The primer of quick detection, it is characterised in that
The gene order of sense primer 5 ' -3 ' is：AAGCACTTGAAGGAGAAGGT；
The gene order of anti-sense primer 5 ' -3 ' is：AAAGCGGAAGAATGTGTCAG.
6. utilize MTHFR-C677T, RS1801133 gene pleiomorphism with primer detection in the kit described in claim 5
The molecular beacon of quick detection, it is characterised in that：
The gene order of the 5 ' of wild type molecular beacon -3 ' is：CGTGCAGAAATCGGCTCCCGCAGTGCACG；
5 ' -3 ' gene order of mutant molecules beacon is：CGCGACGAAATCG+ACTCCCGCAGACGTCGCG；
+ the base modified for lock nucleic acid；
And 5 ' ends of wild type molecular beacon and mutant molecules beacon gene order and 3 ' hold be respectively equipped with fluorophor and with
The quenching group that fluorophor coordinates.
7. the molecular beacon of MTHFR-C677T, RS1801133 gene pleiomorphism quick detection according to claim 6, its
It is characterised by：Described fluorophor is 6-Fam labelling groups, and described quenching group is BHQ1 labelling groups；
Or fluorophor is the labelling groups of Alexa Fluor 594, described quenching group is BHQ2 labelling groups.
8. MTHFR-C677T, RS1801133 for being carried out using the primer described in claim 6 or 7, molecular beacon and kit
Gene pleiomorphism quick determination method, it is characterised in that：Operating method is,
（1）Gargled 2 times with water before sampling, be more than 5 seconds every time, swallowed 2-3 times after gargling；
（2）Cheek wall in the nearly 90 ° of contacts oral cavity in sampling portion of sampler is taken, uniformly scrapes 5 times, is 1 time up and down；
（3）By in the sampling portion insertion PCR reaction solutions of sampler after sampling, mix；
（4）The human genome DNA for taking 1 μ L to purify is added in another PCR reaction solution, is mixed；
（5）The PCR reaction solutions for completing sample-adding are put into quantitative PCR apparatus, run response procedures.
9. MTHFR-C677T, RS1801133 gene carried out using the primer described in claim 8, molecular beacon and kit
Polymorphism quick determination method, it is characterised in that：The response procedures of quantitative PCR apparatus are：
A, pre-degeneration：95 DEG C, 5min, 1 circulations of temperature；
B, it is denatured：95 DEG C, 8s；
C, annealing and extension：55 DEG C, 35s；
D, B and c program operate 50 circulations.
10. MTHFR-C677T, RS1801133 gene pleiomorphism quick determination method as claimed in claim 9, its feature exist
In：Described quantitative PCR apparatus is binary channels, and excitation wavelength is respectively 450-500nm and 550-600nm.
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Address after: 400084 Chongqing, Dadukou District Chunhui Road, 101 Cui Bo Road 4 4 2-1, 2-9 Applicant after: CHONGQING JINGYIN BIOTECHNOLOGY CO., LTD. Address before: 400051 9, LONCIN international office 5, 2 Shiping bridge, Kowloon Po, Chongqing. Applicant before: CHONGQING JINGYIN BIOTECHNOLOGY CO., LTD. |
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Application publication date: 20171208 |